Progress in chemical synthesis of nuclease resistant oligonucleotides (Methods Mol. Biol. (1993) Vol. 20, (Agrawal, ed.) Humana Press, Totowa, N.J.) and developments in large scale solid phase synthesis of oligonucleotides (Agrawal, ed.) Methods Mol. Biol. (1993) Vol. 20, Humana Press, Totowa, N.J.); Padmapriya et al. (1994) Antisense Res. Dev. 4:185-199) has permitted antisense oligonucleotides to advance to human clinical trials (Bayever et al. (1993) Antisense Res. Dev. 3:383-390). In principle, antisense oligonucleotides utilize highly sequence-specific complementary nucleo-base recognition of target nucleic acids through Watson-Crick hydrogen bonding between A and T, and G and C, that leads to the development of less toxic and more site specific chemotherapeutic agents (Stephenson et al. (1978) Proc. Natl. Acad. Sci. (USA) 75:285-288). As per theoretical calculations, an oligonucleotide of 13 or more bases long should bind to a unique sequence that occurs only once in a eucaryotic mRNA pool.
Contrary to the popular belief, it was recently shown that the increase in the length of an antisense oligonucleotide beyond the minimum length that can hybridize to the target (i.e. 11-14 bases) decreases its specificity rather than increasing (Woolf et al. (1992) Proc. Natl. Acad. Sci. (USA) 89:7305-7309). Potentially, this decrease in hybridization specificity would lead to non-sequence-specific target binding and subsequent increased toxicity (Stein et al. (1993) Science 261:1004-1012).
Thus, what is needed are improved antisense oligonucleotides optimized for therapeutic and diagnostic use which have improved affinity, specificity, and biological activity, and little or no toxicity.
The present invention provides cooperative oligonucleotides with improved sequence specificity for a single-stranded target, reduced toxicity, and improved biological activity as antisense molecules.
Surprisingly, it has been discovered that two short oligonucleotides (25 nucleotides or less) bind to adjacent sites on the target nucleic acid in a cooperative manner, allowing for an interaction with greater sequence specificity than can a single longer oligonucleotide having a length equal to the two shorter oligonucleotides.
Accordingly, in a first aspect, the present invention provides a composition including at least two synthetic cooperative oligonucleotides, each comprising a region complementary to one of tandem, non-overlapping regions of a target single-stranded nucleic acid, and a dimerization domain at a terminus of each of the oligonucleotides. The dimerization domains of the cooperative oligonucleotides are complementary to each other, and the target nucleic acid being an mRNA, single-stranded viral DNA, or single-stranded viral RNA.
In some preferred embodiments, the oligonucleotides each are complementary to tandem regions of the target nucleic acid that are separated by 0 to 3 bases. In some preferred embodiments, each of the oligonucleotides are about 9 to 25 nucleotides in length.
In one embodiment, the composition consists of two cooperative oligonucleotides, the dimerization domain of a first or one of the oligonucleotides being located at its 3xe2x80x2 terminal portion, and being complementary to the dimerization domain of a second or the other oligonucleotide which is located at its 5xe2x80x2 terminal portion. Alternatively, the dimerization domain of the first cooperative oligonucleotide is located at its 3xe2x80x2 terminal portion, and is complementary to the dimerization domain of a second oligonucleotide which is located at its 3xe2x80x2 terminal portion. Alternatively, the dimerization domain of the first cooperative oligonucleotide is located at its 5xe2x80x2 terminal portion, and is complementary to a dimerization domain of the second oligonucleotide which is located at its 5xe2x80x2 terminal portion.
The invention provides in another aspect a duplex structure comprising first and second synthetic cooperative oligonucleotides, each oligonucleotide comprising a region complementary to the non-overlapping, tandem regions of the target nucleic acid which is an mRNA, single-stranded viral RNA, or single-stranded viral DNA. The first oligonucleotide in the duplex has a terminal dimerization domain complementary and hybridized to the dimerization domain of the second oligonucleotide. In some embodiments, each of the oligonucleotides are about 9 to 25 nucleotides in length, and in others, the dimerization domains of the first and second oligonucleotides each comprise about 3 to 7 nucleotides. In some embodiments, the invention provides first and second oligonucleotides which are complementary to tandem regions of the target nucleic acid separated by 0 to 3 bases.
The invention also provides pharmaceutical formulations containing the compositions or duplex structures described above, and methods of inhibiting the expression of a nucleic acid in vitro comprising the step of treating the nucleic acid with the pharmaceutical formulations of the invention. In some embodiments, the first and second oligonucleotides are complementary to an HIV DNA or an HIV RNA.
In another aspect, the invention provides a ternary complex comprising the duplex structure of the invention and a target oligonucleotide to which regions of the first and second cooperative oligonucleotides are complementary. The target oligonucleotide is an mRNA, a single-stranded viral DNA, or a single-stranded DNA.